Biology subjects

Morawski, P. A.

Publications and source records attributed to Morawski, P. A..

6 recordsLinked to original sources

bioRxiv2025

The altered T cell landscape in Systemic Sclerosis patients is characterized by dysfunctional type 1 immunity.

BackgroundT cells in patients with chronic autoimmunity show features of dysfunction and exhaustion. However, the functionally-defined T cell subsets affected by these changes remain poorly characterized. Here, we sought to reveal aberrations in the composition, phenotype and function of canonical T cell subsets in the blood of Systemic Sclerosis (SSc) patients, compared to healthy subjects and Systemic Lupus Erythematosus (SLE) patients. MethodsWe developed a novel multidimensional flow-cytometry panel to simultaneously detect lineage-defining transcription factors, co-inhibitory receptors and other functional markers to characterize T cell subsets without in vitro restimulation. We compared T cell landscapes in SSc and SLE patients, and healthy subjects, using Optimized t-SNE and PhenoGraph algorithms. Cytokine production in patient samples was determined using intracellular cytokine staining. Transcriptomic analysis was performed to assess the role of IFN-{gamma} in fibroblasts. ResultsThe data show altered distribution and aberrant functional states of transcription factor-defined T cell subsets in patients with autoimmunity, with shared and unique features between diseases and their subtypes. Strikingly, SSc and SLE patients showed a severe deficiency in subsets of CD8+ and CD4-CD8- T cells that expressed T-bet, which is critical for IFN-{gamma}-dependent type 1 immunity, and exhibited features of exhaustion. Moreover, TIGIT+Foxp3+ regulatory T cells, known to suppress Th1 responses, were selectively increased in SSc. Functionally, T cells from SSc patients produced less IFN-{gamma}, a cytokine that suppresses pro-fibrotic gene expression in fibroblasts. ConclusionOur study demonstrates that SSc patients have a defective IFN-{gamma}-producing T cell compartment that may lead to reduced anti-fibrotic T cell activity, enabling chronic fibrosis development. FundingNational Institute of Allergy and Infectious Diseases/NIH, National Institute of Arthritis and Musculoskeletal Diseases/NIH, National Heart, Lung and Blood Institute/NIH, National Scleroderma Foundation, National Jewish Health

immunology↗

bioRxiv2024

T cells promote distinct transcriptional programs of cutaneous inflammatory disease in human skin structural cells

T cells and structural cells coordinate appropriate inflammatory responses and restoration of barrier integrity following insult. Dysfunctional T cells precipitate skin pathology occurring alongside altered structural cell frequencies and transcriptional states, but to what extent different T cells promote disease-associated changes remains unclear. We show that functionally diverse circulating and skin-resident CD4+CLA+ T cell populations promote distinct transcriptional outcomes in human keratinocytes and fibroblasts associated with inflamed or healthy tissue. We identify Th17 cell-induced genes in keratinocytes that are enriched in psoriasis patient skin and normalized by anti-IL-17 therapy. We also describe a CD103+ skin-resident T cell-induced transcriptional module enriched in healthy controls that is diminished during psoriasis and scleroderma and show that CD103+ T cell frequencies are altered during disease. Interrogating clinical data using immune-dependent transcriptional signatures defines the T cell subsets and genes distinguishing inflamed from healthy skin and allows investigation of heterogeneous patient responses to biologic therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/606077v4_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@fb573corg.highwire.dtl.DTLVardef@11c4eb3org.highwire.dtl.DTLVardef@17304cforg.highwire.dtl.DTLVardef@40d6ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Chromatin conformation dynamics during CD4+ T cell activation implicates autoimmune disease-associated genes and regulatory elements

Genome-wide association studies (GWAS) have identified hundreds of genetic signals associated with autoimmune disease. The majority of these signals are located in non-coding regions and likely impact cis-regulatory elements (cRE). Because cRE function is dynamic across cell types and states, profiling the epigenetic status of cRE across physiological processes is necessary to characterize the molecular mechanisms by which autoimmune variants contribute to disease risk. We localized risk variants from 15 autoimmune GWAS to cRE active during TCR-CD28 costimulation of naive human CD4+ T cells. To characterize how dynamic changes in gene expression correlate with cRE activity, we measured transcript levels, chromatin accessibility, and promoter-cRE contacts across three phases of naive CD4+ T cell activation using RNA-seq, ATAC-seq, and HiC. We identified [~]1,200 protein-coding genes physically connected to accessible disease-associated variants at 423 GWAS signals, at least one-third of which are dynamically regulated by activation. From these maps, we functionally validated a novel stretch of evolutionarily conserved intergenic enhancers whose activity is required for activation-induced IL2 gene expression in human and mouse, and is influenced by autoimmune-associated genetic variation. The set of genes implicated by this approach are enriched for genes controlling CD4+ T cell function and genes involved in human inborn errors of immunity, and we pharmacologically validated eight implicated genes as novel regulators of T cell activation. These studies directly show how autoimmune variants and the genes they regulate influence processes involved in CD4+ T cell proliferation and activation.

immunology↗

bioRxiv2020

Designed proteins assemble antibodies into modular nanocages

Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by -SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.

biochemistry↗

bioRxiv2019

Rapid light-dependent degradation of fluorescent dyes in chemically-defined media

Chemically defined serum-free media is increasingly used as a tool to help standardize experiments by eliminating the potential variability contributed by pooled serum. These media are formulated for the culture and expansion of specific cell types, maintaining cell viability without the need for exogenous animal proteins. Formulated serum-free media could thus help improve viability and reduce variability during sample preparation for flow cytometry, yet a thorough analysis of how such media impact fluorochrome-antibody conjugates has not been performed. In this study, we expose fluorescent antibody-labeled cells or antibody capture beads to white light in the presence of various hematopoietic cell culture media and provide evidence that formulated serum-free media permit rapid light-initiated fluorescent dye degradation in a cell-independent manner. We observed fluorescence signal loss of several dyes, which included fluorescence spillover into adjacent detectors. Finally, photostability of antibody-fluorochrome conjugates in formulated serum-free media is partially restored in the presence of either serum or vitamin C, implicating reactive oxygen species in the observed signal loss. Thus, our data indicate that formulated serum-free media designed to standardize cell culture are not currently optimized for use with fluorochrome-antibody conjugates, and thus extreme caution should be exercised when using these media in cytometric experiments.

cell biology↗

bioRxiv2018

Continual exit of human cutaneous resident memory CD4 T cells that seed distant skin sites

Tissue-resident memory T cells (TRM) persist locally in non-lymphoid tissues where they provide front-line defense against recurring insults. TRM at barrier surfaces express the markers CD103 and/or CD69 which function to retain them in epithelial tissues. In humans, neither the long-term migratory behavior of TRM nor their ability to re-enter the circulation and potentially migrate to distant tissue sites have been investigated. Using tissue explant cultures, we found that CD4+CD69+CD103+ TRM in human skin can downregulate CD69 and exit the tissue. Additionally, we identified a skin-tropic CD4+CD69-CD103+ population in human lymph and blood that is transcriptionally, functionally and clonally related to the CD4+CD69+CD103+ TRM population in the skin. Using a skin xenograft model, we confirmed that a fraction of the human cutaneous CD4+CD103+ TRM population can re-enter circulation, and migrate to secondary human skin sites where they re-assume a TRM phenotype. Thus, our data challenge current concepts regarding the strict tissue compartmentalization of CD4+ T cell memory in humans. One Sentence SummaryHuman CD4+CD103+ cutaneous resident memory T cells are found in the circulation of healthy subjects, and these cells can seed distant skin sites.

immunology↗